Funai Katsuhiko, Cartee Gregory D
Muscle Biology Laboratory, School of Kinesiology, University of Michigan, Ann Arbor, Michigan, USA.
Diabetes. 2009 May;58(5):1096-104. doi: 10.2337/db08-1477. Epub 2009 Feb 10.
Phosphorylation of two members of the TBC1 domain family of proteins, Akt substrate of 160 kDa (AS160, also known as TBC1D4) and TBC1D1, has been implicated in the regulation of glucose transport in skeletal muscle. Insulin-stimulated phosphorylation (measured using the phospho-Akt substrate [PAS] antibody) of AS160 and TBC1D1 appears to occur in an Akt-dependent manner, but the kinases responsible for contraction-stimulated PAS-AS160 and PAS-TBC1D1 remain unclear. AMP-activated protein kinase (AMPK) and Akt, both activated by contraction, can each phosphorylate AS160 and TBC1D1 in cell-free assays.
To evaluate the roles of AMPK and Akt on insulin- or contraction-stimulated PAS-AS160, PAS-TBC1D1, and glucose transport, rat epitrochlearis was incubated with and without compound C (inhibitor of AMPK) or Wortmannin (inhibitor of phosphatidylinositol [PI] 3-kinase, which is upstream of Akt) before and during insulin stimulation or contraction.
Insulin-stimulated glucose transport and phosphorylation of both AS160 and TBC1D1 were completely inhibited by Wortmannin. Wortmannin eliminated contraction stimulation of phospho-Ser(21/9)glycogen synthase kinase 3alpha/beta (pGSK3; Akt substrate) and PAS-AS160 but did not significantly alter pAMPK, phospho-Ser79acetyl CoA carboxylase (pACC; AMPK substrate), PAS-TBC1D1, or glucose transport in contraction-stimulated muscle. Compound C completely inhibited contraction-stimulated pACC and PAS-TBC1D1 and partially blocked glucose transport, but it did not significantly alter pAkt, pGSK3, or PAS-AS160.
These data suggest that 1) insulin stimulates glucose transport and phosphorylation of AS160 and TBC1D1 in a PI 3-kinase/Akt-dependent manner, 2) contraction stimulates PAS-AS160 (but not PAS-TBC1D1 or glucose transport) in a PI 3-kinase/Akt-dependent manner, and 3) contraction stimulates PAS-TBC1D1 and glucose transport (but not PAS-AS160) in an AMPK-dependent manner.
TBC1结构域蛋白家族的两个成员，160 kDa的Akt底物（AS160，也称为TBC1D4）和TBC1D1的磷酸化，与骨骼肌中葡萄糖转运的调节有关。胰岛素刺激下AS160和TBC1D1的磷酸化（使用磷酸化Akt底物[PAS]抗体检测）似乎以Akt依赖的方式发生，但负责收缩刺激的PAS-AS160和PAS-TBC1D1的激酶仍不清楚。AMP激活的蛋白激酶（AMPK）和Akt都可被收缩激活，在无细胞试验中它们均可使AS160和TBC1D1磷酸化。
为评估AMPK和Akt在胰岛素或收缩刺激的PAS-AS160、PAS-TBC1D1及葡萄糖转运中的作用，在胰岛素刺激或收缩之前及期间，将大鼠肱三头肌与化合物C（AMPK抑制剂）或渥曼青霉素（磷脂酰肌醇[PI] 3激酶抑制剂，PI 3激酶在Akt上游）一起或不与它们一起孵育。
渥曼青霉素完全抑制胰岛素刺激的葡萄糖转运以及AS160和TBC1D1的磷酸化。渥曼青霉素消除了收缩对磷酸化丝氨酸（21/9）糖原合酶激酶3α/β（pGSK3；Akt底物）和PAS-AS160的刺激作用，但对收缩刺激的肌肉中的pAMPK、磷酸化丝氨酸79乙酰辅酶A羧化酶（pACC；AMPK底物）、PAS-TBC1D1或葡萄糖转运没有显著影响。化合物C完全抑制收缩刺激的pACC和PAS-TBC1D1，并部分阻断葡萄糖转运，但对pAkt、pGSK3或PAS-AS160没有显著影响。
这些数据表明：1）胰岛素以PI 3激酶/Akt依赖的方式刺激葡萄糖转运以及AS160和TBC1D1的磷酸化；2）收缩以PI 3激酶/Akt依赖的方式刺激PAS-AS160（但不刺激PAS-TBC1D1或葡萄糖转运）；3）收缩以AMPK依赖的方式刺激PAS-TBC1D1和葡萄糖转运（但不刺激PAS-AS160）。
